Circulating cell-free nucleic acid fragments, usually shorter than 1000 bp, such as tumor-specific extracellular nucleic acid fragments in the blood or fetal nucleic acids in maternal blood can be isolated from serum or plasma. (Lo et al., Lancet 350:485-487 (1997); Chan et al., Clin. Chem. 52:2211-2218 (2006)) Analysis of circulating cell-free nucleic acid fragments can be useful in characterizing certain cancers (Murtaza et al., Nature 497(7447):108-12 (2013)) and disease states (Swarup et al., FEBS Lett. 581(5):795-9 (2007)) as well as in fetal genetic analysis (Lo et al., supra; Chiu et al., Clin. Chem. 47:1607-1613 (2001); Chan et al., supra). The concentrations of circulating cell-free nucleic acid in biological fluids such as plasma, serum, or urine can vary considerably (˜50 pg/ml to >100 ng/ml) and depend on the individual, disease state, therapeutic regimen, and period of gestation (Swarup et al., supra). For example, the concentrations of circulating cell-free fetal DNA has been shown to be 3.4% (range 0.39%-11.9%) and 6.2% (range 2.33%-11.4%) of the total plasma DNA in early and late pregnancy, respectively. (Lo et al., Am. J. Hum. Genet. 62(4):768-775 (1998)).
There are a number of known methods of purifying single- and double-stranded NA contained in biological fluids such as human blood, serum, and cultured cells, as well as plants, animal and human tissues, and other specimens. However, such methods can result in very low yields and do not always work well when trying to extract small amounts of nucleic acids from large samples, such as the relatively small amounts of cell-free nucleic acids present in biological fluids. Some methods entail use of chaotropes which facilitate dissociation of cell-free nucleic acid from proteins. As known in the art, higher concentrations of chaotrope are desirable for release of nucleic acids from proteins and capture on a solid phase such as a silicon phase. However, there are disadvantages associated with use of high concentration or molarity of chaotropes. The high molarity of chaotropes may in turn result in viscous solutions which may be difficult to work with. High molarities of chaotropes also necessitate diluting the biological fluid sample several-fold causing undesirably high fluid volumes, which are not amenable for use with existing well established automation equipment. For example, the QIAamp® Circulating Nucleic Acid kit, commercially available from Qiagen, entails about a four-fold dilution of the bodily fluid and use of carrier RNA.